M olecular Clon ing of D eletion Br idge Fragm en t of Y unnanese
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- Πλάτων Δημητρακόπουλος
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1 V o l 15,N o 1 Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy Feb 1999 Y unnanese ( A Χ Β) 0-3 (,,, ) Yunnanese ( A Χ Β) 0 2, 3, EM BL 3, 11 G Χ 617 kb 1115 kb 3 ( ) 1115 kb Β kb DNA, 5 A Χ L 1, Β 66 kb, Ch inese ( A Χ Β) kb E cor g 413 bp 5 3 L 1 3 ( A Χ Β) 0 2 M olecular Clon ing of D eletion Br idge Fragm en t of Y unnanese ( A Χ Β) 0 - tha la ssem ia and Sequence Ana lysis of the D eletion Junction Reg ion ZHAN G Junw u, ZHAN G Xueqing, ZHAO Yan jun (N ational L aboratory of M ed ical M olecu lar B iology, T he Institu te of B asic M ed ical S ciences, P ek ing U nion M ed ical Colleg e and Ch inese A cad emy of M ed ical S ciences, B eij ing ) Abstract In o rder to study the m echan ism s leading to the Yunnanese ( A Χ Β) 0 2thalassem ia dele2 tion characterized p reviou sly and search fo r enhancer2like sequence in the jux tapo sed region from the 3 end of the Β2glob in gene clu ster, a genom ic lib rary of a heterozygo te w ith the deletion has been con structed u sing Κphage vecto r EM BL 3 A clone con tain ing the deletion b ridge fragm en t w as iden tified, w h ich con sisted of a 6 7 kb of the G Χglob in gene region and a 11 5 kb of 3 jux ta2 po sed sequence that w as located no rm ally in abou t kb dow n stream from the Β2glob in gene A fine physical m ap of th is 11 5 kb region w as m ade By sequence analysis it w as show n that the 5 b reakpo in t of the Yunnanese ( A Χ Β) 0 2thalassem ia deletion w as located betw een2116 and 2117 bp of the A Χglob in gene, w h ile the 3 b reakpo in t occu rred w ith in a L 1 fam ily sequence and at 413 bp 5 of the E cor g site that lay 12 2 kb up stream of the 3 b reakpo in t of the Ch inese ( A Χ Β) 0 2tha2 lassem ia deletion N o obviou s hom o logy of the region s su rrounding the 5 and 3 b reakpo in ts w as detected, suggesting that the Yunnanese ( A Χ Β) 0 2thalassem ia deletion had resu lted from a non2 hom o logou s recom b ination even t that w as p robab ly m ediated by an L 1 elem en ṫ Key words: ( A Χ Β) 0 2T halassem ia, D eletion junction region,n on2hom o logou s recom b ination 3 ( ) :,, T el: (010) , Fax: (010) : , :
2 1 : Yunnanese ( A Χ Β) Β 11, Am ersham 5 (5 Ε2 G Χ A Χ2 2 p 500H D 1M ager Β 3 ), A Χ5 118 kb pt Kneo A Χ, 6 Ε 112 D NA D NA Χ, EBV Χ Β DNA [ 3 ] 100 Λg DNA B g lg, 2, Β T E DNA (8% 40% ), Χ Beckm an SW rgm in h 0 75 m l, ( Β) 0 2 [ ( A Χ Β) 0 2 ], (H PFH ) 113 (H bf),, DNA, A Χ H bf,, 313 kb DNA, [ 1 ] 1815 kb DNA kb DNA 2 DNA G Χ Yunnanese ( A Χ Β) D NA [ 2 ] H bf 9% 17%, 10 Λl : 1 Λg EM BL 3, 017 g, (H b 1017 B g l g, 10 mm o lgl T ris2hc l g ) 90 kb (ph 716), 10 mm o lgm gc l2, 1 mm o lgl A T P 1 DNA, 5 A Χ 011 DNA 20 3 h, 50 Λl 012 kb, 3 Β 66 kb, 20 2 h 0 5 m l SM (0 1 DNA, m o lgl N ac l, 8 mm o lgl M gso 4, 5 mm o lgl T ris G Χ HC l[ph 7 5 ], 0 01% ) 25 Λl, G [ 4 Χ 4 ], pfugm l, 3 DNA, DNA 10 6 pfu Ep stein2barr (EBV ) Yun h nanese ( A Χ Β) 0 [ 3 2 B ] 32 P 5 A Χ (118 kb EM BL 3 B am H g B am H g 2H ind g ) p 500H ( Β KW kb 0 5 kb H indg P rom ega T 4 DNA Boeh ringer M annheim (Seque2 nase V ersion 2 0) U n ited States B io2 chem ical Α2 32 P2dCT P Α2 32 P2dA T P, 5 Λl 6% 115 [ 4 ] KW 251, mm LB pfu Κ D NA ) [ 5 ] [ 4 ] Κ
3 DNA, 117 D NA, 117 kb H ind g DNA pgem 24z DNA pfu, Λg DNA 20 Λl 2 m o lgl 15 m in 500 m l LB, h N aoh 22 mm o lgl ED TA 5 m in, 3, 10 m l Λl 3 m o lgl (ph 5 2) 10 m in (Sequenase V ersion 2 0) DN aseg RN ase 1 Λggm l, 30 m in N ac l 1 m o lgl, 1 h rgm in 10 m in g (PEG8 000), kb B g l 1 h rgm in 10 m in, 8 m l SM, ( pfugλg EM BL 3 ), 32 P A Χ5 000 rgm in 4 2 h 118 kb ( G Χ ) 2 m l SM [4 ] p 500H, DNA,, DNA, A Χ5 p 500H 2 g DNA EM BL 3,, 5 F ig 1 A nalysis of ΚYBg DNA restriction fragm ents (The DNA w ere digested w ith restriction endonucleases and separated electropho resly undergo ing agaro se gel The DNA fragm ents w ere analyzed by Southern blo t hybridization w ith A Χ5 118 kb p robe and p500h p robe respectively A : A ccg ; B: B am H g ; E: E cor g ; H: H p ag ; N : N cog ; S: S alg ; X: X bag ; Κ: ΚDNA gh indg m arker
4 1 : Yunnanese ( A Χ Β) DNA DNA, Sa Ig kb DNA G Χ, kb DNA, 1115 kb 3 kb, 1115 kb Β A Χ5 p 500H kb DNA DNA, ΚYBg G Χ 3 18 kb, ΚYBg ΚYBg DNA 6, A Χ5 DNA Sou thern p 500H Sou thern,,, DNA ( F ig 1 [ 3 ]), DNA 1115 kb, F ig 2 F ig 2 Physical m app ing of the DNA region dow nstream from the 3 breakpo int of the Yunnanese ( A Χ Β) 0 2thalassem ia deletion T he restriction enzym e sites w ere determ ined by m app ing ΚYBg and no rm al genom ic DNA Yun, Yunnanese; 5 b: the 5 breakpo int; 3 b: the 3 breakpo int; A : A ccg ; Ba, B am H g ; Bg: B g lg ; E: E cor g ; H: H indg ; N : N cog ; S: S acg ; X: X ba g 213 D NA Β 66 kb 500 bp DNA, ( A Χ Β) kb E cor g, 413 bp Β DNA 3 DNA 5 [ 6 ], 5 A Χ DNA, (A ) (G) (F ig 3) 3 Yunnanese ( A Χ Β) 0 2 F ig 3 A comparison of the DNA sequence bo rdering the deletion junction po int of Yunnanese ( A Χ Β) 0 2thalassem ia w ith the no rm al sequence surrounding the 5 breakpo int T he deletion junction is indicated by an arrow Po sition indicated is relative to the cap site of the A Χglobin gene Sequence homo logy is indicated by vertical dashes Two CCTTG direct repeats are underlined
5 bp, L 1 L 1 (AM L ) D EK23 CAN, 3 CAN L 1 DNA D EK L 1 EM BL 3, [ 11 ], L 1 (Ε pfugλg EM BL 3 ), Yunnanese ( A Χ Β) 0 2 DNA, G Χ ( pfugm l EM BL 3,, ) DNA References, DNA ( 2 ) 5 3 DNA, Yunnanese ( A Χ Β) 0 2, [ 7 DNA ] 5 CCT T G, [ 7 ] DNA Yunnanese ( A Χ Β) A Χ, 3 L 1 Β, 5 Β, ( A lu L 1 ) [ 8 ], 3 L 1 3 Β kb L 1 8 Gilm an J G, B rinson E C,M ish im a N T he 32 6 kb lndian ( Β) 0 2 L ao tian ( Β) 0 2 T hai ( Β) 0 2 Scilian ( Β) 0 2 B lack ( Β) 0 2 East Eu ropean ( Β) 0 2 ltalian H PFH 2 5 [ 1 ] 611 kb L 1 1 kb 314 kb L 1 [ 8 ], 3 indian ( Β) 0 2 [ 8 ] T u rk ish ( A Χ Β) 0 2 [ 9 ] B lack ( A Χ Β) 0 [ 1 2 ] L 1 DNA ( long in terpersed elem en ts, L IN ES L 1) L 1 6 kb, 4 000, 5 2 L 1 DNA 1 Stam atoyannopoulo s G, N ienhuis A W H em go lobin sw itch ing In: Stam atoyannopoulo s G, N ienhuis AW, L eder P, M ajerus P W eds T he M olecular B asis of B lood d iseaseṡ Ph iladelph ia: P A Saundes, 1994: Zhang J W, Song W F, Zhao Y J,W u G Y, Q iu ZM,W ang F N, Chen S S Stam atoyannopoulo s G M o lecular characterization of a novel fo rm of ( A Χ Β) 0 2thalassem ia deletion in a Ch inese fam i2 ly B lood, 1993, 81: Zhang J W ( ), Q iao J ( ), Song W F ( ), Q iu Z M ( ) A ctive exp ression of G Χ globin gene on ch romo2 som e 11 w ith Yuunanese ( A Χ Β) 0 2thalassem ia deletion in M EL cells S cience in Ch ina (Series C) 1996, 39: Sam brook J, F ritsch E F, M aniatis T M olecu lar C loning: A L aboratory M anual, 2nd ed N ew Yo rk: Co ld Sp ring H arbo r L abo rato ry P ress, M ager D L, H entho rn P S, Sm ith ies O A Ch inese G Χ + ( A Χ Β) 0 2 thalassem ia deletion: comparison to o ther deletions in the hu2 m an Β2globin gene cluster and sequence analysis of the break2 po ints N ucleic A cid s R es, 1985, 13: B ilofsky H S, Burk s C, F ickett J W, Goad W B, L ew itter F I, R indone W P, Sw indell C D, Fung C S T he GenBank genetic se2 quence databank N ucleic A cid s R es, 1986, 14: H entho rn P S, Sm ith is O, M ager D L M o lecular analysis of deletion junctions and locations of breakpo ints Genom ics, 1990, 6: thalassem ia deletion ends in a 3 4 kb L 1 elem ent dow nstream of the Β2globin gene B r J H aem atol, 1992, 82: : T rent R J, Jones R W, C legg J B,W eatherall D J, D avidson R, W ood W G ( A Χ Β) 0 2thalassem ia: sim ilarity of pheno type in four different mo lecular defeats, including one new ly described B r J H aem atol, 1984, 57: H entho rn P S, Sm ith is O, N akatsuji T, Felice A E, Gardiner M B, Reese A L, H uism an T H J ( A Χ Β) 0 2thalassem ia in black s is due to a deletion of 34 kb of DNA B r J H aem atol, 1985: 59: von L indern M, B reem s D, van Baal S, A driaansen H, Gro sveld G Characterization of the translocation breakpo int sequences of two D EK2CAN fusion genes p resent in t (6, 9) acute m yelo id leukem ia and a SET 2CAN fusion gene found in a case of acute undifferentiated leukem ia Genes Ch rom Cancer, 1992, 5:
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